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Abstract:
Growing research has implicated Fipronil as a teratogen in the development of chordates. Fipronil is a pesticide that acts to disrupt and inhibit normal nerve activity. The result is excessive nerve stimulation caused by overstimulation of gamman-aminobuytric (GABA) gated chloride ion channels. Experiments using zebrafish (Danio rerio) indicate Fipronil is teratogenic at concentrations above 0.7 μM. In this study, the effect of increasing concentrations (0.7 μM, 1.1 μM, 2.3 μM) of Fipronil on neurological development were examined. Our results showed that within six hours of GABA-receptor expression (36 hours-high pec) there was neurological damage as indicated by an abnormal behavioral escape touch response (resembling the accordion class phenotype), abnormal morphology (ventrally curved long fin), and damage to neurological tissue (notochord damage concentrated in the dorsal area of the trunk-tail interface). Within 48 hours (long-pec) damage was more extreme as observed in behavior, long tail morphology, and damage to neurological tissue. Furthermore, distinct regions of necrotic tissue were visible, accompanied by aberrant circulation. This data demonstrates Fipronil teratogenicity increases with exposure and concentration and is correlated with increasing damage to the notochord during development. This vertebrate study may have implications for diseases such as fetal alcohol syndrome (FAS) and autism on the phenotypic impact of Fipronil on the development of GABAergic neural pathways.

The Teratogenic Effects of Fipronil on the Neurological Development of Zebrafish Embryos Joshua E. Mendoza-Elias§ Biology 205L: Experiments in Developmental Biology and Molecular Genetics Fall 2007 Duke University Dr. Alyssa Perz-Edwards, Ph.D. Trinity School of Arts and Sciences, Biology Department Submitted: Wednesday, December 13, 2006 Abstract Growing research has implicated Fipronil as a teratogen in the development of chordates. Fipronil is a pesticide that acts to disrupt and inhibit normal nerve activity. The result is excessive nerve stimulation caused by overstimulation of gamman-aminobuytric (GABA) gated chloride ion channels. Experiments using zebrafish (Danio rerio) indicate Fipronil is teratogenic at concentrations above 0.7 µM. In this study, the effect of increasing concentrations (0.7 µM, 1.1 µM, 2.3 µM) of Fipronil on neurological development were examined. Our results showed that within six hours of GABA-receptor expression (36 hours- high pec) there was neurological damage as indicated by an abnormal behavioral escape touch response (resembling the accordion class phenotype), abnormal morphology (ventrally curved long fin), and damage to neurological tissue (notochord damage concentrated in the dorsal area of the trunk-tail interface). Within 48 hours (long-pec) damage was more extreme as observed in behavior, long tail morphology, and damage to neurological tissue. Furthermore, distinct regions of necrotic tissue were visible, accompanied by aberrant circulation. This data demonstrates Fipronil teratogenicity increases with exposure and concentration and is correlated with increasing damage to the notochord during development. This vertebrate study may have implications for diseases such as fetal alcohol syndrome (FAS) and autism on the phenotypic impact of Fipronil on the development of GABAergic neural pathways. Keywords: zebrafish, fipronil, teratogen, GABA channels, notochord, necrosis. ________________________________________________________________________ Joshua Mendoza-Elias, Trinity College of Arts and Sciences, Department of Biology. § E-mail: joshua.mendozaelias@duke.edu. Website: http://www.duke.edu/~jme17

Mendoza-Elias, Joshua E. Teratogenic Effects of Fipronil on Zebrafish Neurological Development 1 Introduction Fipronil is a commercial pesticide designed to kill pests (fleas, cockroaches, ants, termites). Common usage includes agricultural pesticide and a topical application for household pets. Fipronil functions by disrupting insect gamma-aminobutyric acid (GABA) – gated chloride channels by blocking the inhibitory action of GABA receptors in the central nervous system (CNS) [1, 2, 3]. The result is disruption of GABA inhibitory action. Physiologically, fipronil exposure causes hyperexcitation at low doses and death at high doses. More importantly, insect and vertebrate GABA receptors have common structural features as they belong to the ligand-gated ion channel superfamily [5]. However, fipronil has a higher binding affinity for insect GABA receptors than vertebrate GABA receptors [6]. This physical property is believed to account for the lower toxicity of Fipronil in mammals [5]. The result is excessive nerve stimulation caused by overstimulation of gamman-aminobutyric (GABA) gated chloride ion channels [1, 2, 4, 6]. However, experiments using the zebrafish (Danio rerio) indicate that it is a teratogen when concentrations are above 0.07 µM [7]. In light of this, commercial uses of Fipronil have demonstrated that pollution of water can impact non-target aquatic life [8, 9]. This indicates that organisms that are exposed to water contaminated with fipronil may suffer neurotoxicological effects [9]. The minimum concentration (“toxicity floor”) causing abnormality in zebrafish is 0.7 µM. However, concentrations as high as 5.3 µg/l (Cary et al) have been observed [16]. This data implies toxic effects are associated with fipronil and need to be further characterized in vertebrate models. Using the zebrafish model, the effects of increasing concentrations of Fipronil on neurological development will be examined. The results of the experiment will show that within six hours of GABA-receptor expression (36 hours-high pec) there is neurological damage as indicated by an abnormal behavioral response (accordion class phenotype) (see: Movie 1), and an abnormal morphology in the tail. Within 48 hours (long-pec), the damage to neural tissue is more extreme and results in areas of necrotic tissue and disrupted circulation. Loss of behavioral touch- response (stimulation of the Mauthner neuron) was shown to coincide with a rise in fipronil concentration. Brightfield microscopy demonstrated damage to the long-fin morphology in the form of shortening of the rostral-caudal body length as well as necrotic lesions and aberrant systemic blood flow. Preliminary acridine orange stainings supported that the regions of necrotic tissue coincided with cell death. These data show the anterior end of the notochord is the Movie 1: Accordion Class Phenotype Touch Escape Response. Zebrafish treated with 3.0 µM Fipronil to generate the accordion class focal neural damage. phenotype. Brightfield microscopy 20x. Click image to play. Video loops 3 times Furthermore, this data full speed, 1 time 20% speed. 3 tones indicate: beginning of clip, repeated clips, suggests fipronil acts by a slow-motion clips, end of video file. unique mechanism [7]. Page 3 of 13

Teratogenic Effects of Fipronil on Zebrafish Neurological Devlopment Mendoza-Elias, Joshua E. 2 Materials and Methods 2.1 Zebrafish husbandry Wild-type zebrafish (AB strain) were maintained according to standard husbandry procedures [10] at 26° C on a light-dark (14-10 hour) cycle. Artificial system water (egg water) was prepared by mixing purified water with Instant Ocean Sea Salt (Aquatic Ecosystems Inc., Apopka, FL). System water pH was maintained between 7.0-7.4. Adult male and female zebrafish spawned using conventional procedures [12]. Fertilized eggs were collected, cleaned and staged [11] and then transferred to plastic Petri dishes containing fresh system water. 2.2 Chemicals Fipronil (5- amino-1-[2,6-dichloro-4-(trifluromethyl)phenyl]-4- [(trifluromethyl)sulfinyl]-1H-pyrazole) (C12H4Cl2F6N4OS) (98.2% purity) was dissolved in acetone and stock solution of 1.1 µM and 11 µM was prepared in system water with a final acetone concentration of 0.1%. Stocks were stored at 4° C in the dark. Dilutions were prepared daily from stock solutions. PTU – egg water was made by dissolving 1-phenyl-2-thiourea to 0.003% with egg water. This resulted in the blockage of the melanin biosynthetic pathway and loss of pigmentation in melanophores resulting in greater visibility. 2.3 Fipronil Treatments See Biology 205L protocol [12]. Zebrafish embryos were collected and exposed to three concentrations of Fipronil: 0.1% acetone (control), 0.7 µM, 1.1 µM, and 2.3 µM dissolved in PTU-egg water. Each exposure contained 10 embryos. Exposures were initiated before 30 hours post fertilization (hpf). The embryos were then dechorionated at 34 hours post fertilization (hpf). 2.4 Morphological and Behavioral Analysis Two time points were observed: 36 hrs of development (high-pec) and 48 hours (long-fin). Embryos were examined using brightfield microscopy for live video and micrographs. Data on touch response, notochord morphology and circulation were collected. Video was collected at 20x magnification. Three different tones indicate: beginning of video, repeated clips, slow motion clips and end of video file. Notochord morphology was recorded at 100x magnification. Digital light micrographs were obtained using SPOT RT cooled CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Animals were anesthetized with 0.016 mg/ml tricaine and immobilized with 3% methyl cellulose for imaging as necessary [12]. The response to mechanical stimulus (touch) was used as a measure of sensorimotor integration [13]. Dechorionated fish were gently touched on the head with a probe (needle). Escape response was then compared to control. 2.5 Acridine Orange Staining for Necrosis See Biology 205L protocol [12]. Acridine orange staining was then used to examine lesions of necrotized cells in the long fin. Embryos were soaked in 5 µg/ml Acridine Orange for 30 minutes. Embryos were then washed 3 times in egg water and imaged using SPOT RT. Page 4 of 13

Mendoza-Elias, Joshua E. Teratogenic Effects of Fipronil on Zebrafish Neurological Development 5 Discussion The teratogenic effects of fipronil on zebrafish embryos included a decrease of rostral-caudal body length, an impaired motor response, notochord degeneration, necrosis of the anterior notochord and an abnormal circulatory system in damaged regions of the notochord. These defects are found to arise at 30 hpf when GABA receptors are expressed and coincide with the development of the touch-mediated response and the capacity of sustained swimming movements [14]. The reduced fipronil-exposed embryos reflected damage consistent with sustained bilateral contractions resulting in notochord degeneration. Although the developmental defects caused by fipronil are consistent with effects on neuromuscular physiology, the precise targets of fipronil remain uncharacterized. As with fipronil exposure, accordion class mutants have disorganized axial muscle fibers and notochord degeneration. This has been shown to be prevented in fipronil treated zebrafish embryos and accordion class mutants by the pharmacological paralytic MS-222 during development [7]. Fipronil is designed as a selective antagonist of GABA receptors. As a member of the ligand-gated ion channel superfamily, this suggests that the activity of fipronil may affect other targets that possess homologous sequences and/or structures [15]. Evidence for this is supported by treatment with the potent GABAA receptor antagonist, gabazine. The phenotype associated with gabazine exposure does not mimic the effects of fipronil exposure in zebrafish embryos [7]. Experiments with the GlyR antagonist strychnine produced a phenotype almost identical to the phenotype associated with fipronil exposure [7]. The target of these treatments, combined with the accordion phenotype, suggests that affected loci of the accordion class are potential targets for the site of fipronil action. More specifically, the data point to the glycinergic reciprocal inhibitory pathway in the hindbrain and spinal cord [7] as a likely place to search for fipronil targets of action. In studies on prenatal mice, glycine mediated inhibition resulted in reorganization of motor neurons [16]. In fipronil treatments, zebrafish displayed signs of abnormal morphology; however, given that fipronil treated zebrafish can be rescued by removing them from fipronil exposure. Combined with the data, this seems to suggest that damage to the notochord may be due to mechanical stress from the uncontrolled depolarization of GABA receptors. The two wave-like motions from opposite ends of the zebrafish embryos appear to interfere constructively resulting in damage to the anterior region of the dorsal notochord. Currently, the effects between the CNS and the peripheral nervous system (PNS) at the site of notochord damage cannot be determined as coming from either system. But, data and literature implicate the source damage to the zebrafish stems from the disruption of GABA and glycine receptors. This is supported from sequence and similarity among member of the ligand-gated ion channel superfamily [7, 17]. As such, cross reactions are selective between both types of receptors is expected and probably responsible for the fipronil phenotype [17]. 6 Acknowledgements This study was supported by the Biology 205L course (Experiments in Developmental Biology and Molecular Genetics) and a grant from the Howard Hughes Institute. I would like to thank Dr. Alyssa Perz-Edwards, Dr. Nicole Roy, Dr. Amy Bejsovec, Wendy Beane, and Monica Zhang for all their technical expertise and assistance. Page 11 of 13

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